PROJECT SUMMARY HCC is the 3rd most deadly malignancy in the world due to limited success in early and effective therapeutic interventions. Behavior of HCC cells is predicated by tumor microenvironment (TME) which comprises non-cellular and cellular components. One of tumor-associated cell types implicated in promoting HCC is the hepatic stellate cell (HSC) which produces ECM components, matricellular proteins and soluble factors to support HCC growth. Our research into the molecular mechanisms of HSC activation, revealed a novel positive loop of canonical Wnt pathway mediated by stearoyl Co-A desaturase (SCD). This loop involves sequential steps of β-catenin (CTNNB1) amplifying SREBP1c- mediated transcription of Scd2; SCD2 raising cytosolic monounsaturated fatty acids which impair nuclear transport of the mRNA binding protein HuR; and increased cytosolic HuR stabilizing Lrp5/6 mRNA and upregulating their proteins, the functional Wnt co-receptors. Disruption of this loop by conditional Scd2 knockout in activated HSCs (aHSCs) in Scd2f/f;Col1a1Cre mice, attenuates cholestatic and hepatotoxic liver fibrosis, and remarkably live tumor development. This tumor suppression is accompanied by global repression of CTNNB1 and YAP1 in TME, suggesting SCD-dependent release of factor(s) by aHSCs to achieve tumor promotion via global CTNNB and YAP1 upregulation. In search of such factors by combinatorial transcriptomic and lipidomic analyses, we identified 12-HHTrE (12- hydroxyheptadecatrienoic acid), the highest affinity lipid ligand for leukotriene B4 receptor 2 (LTB4R2) as a putative driver for the novel 12-HHTrE-LTB4R2 tumor promotion pathway. 12-HHTrE is a major eicosanoid metabolite released by murine primary aHSCs and human aHSC line LX2 cells. It is enriched in wild type but not in Scd2ff/Col1a1Cre mouse liver TME. It induces via LTB4R2: 1) p-ERK1/2, p(S9)GSK3β, and nuclear CTNNB1; 2) CTNNB1-induced YAP1 transcription via a TCF site in its first intronic enhancer; and 3) nuclear YAP1 and YAP1-target genes in liver cancer cells. Single cell RNA sequencing of mouse TME identifies a unique aHSC subpopulation which co-expresses both HSC marker Lrat and fibroblast marker Fbln2, and selectively expresses Cyp1b1 but not other 12-HHTrE biosynthetic genes. SCD or CTNNB1 knockdown (KD) reduces CYP1B1 expression and 12-HHTrE release by aHSCs in culture. Finally, AAV8-mediated LTB4R2 KD in wild type mice, reproduces tumor suppression observed in Scd2f/f;Col1a1Cre mice. Based on these findings, we hypothesize that the most critical upstream event in the newly disclosed LTB4R2 tumor promotion pathway, is increased12-HHTrE generation by CYP1B1 upregulated in tumor- associated Lrat+Fbln2+ aHSCs and that this subpopulation is a plausible target for anti-HCC therapy. To test this central hypothesis, we aim to determine: 1) if an emergence of Lrat+Fbln2+Cyp1b1+ aHSC subpopulation is prevented by conditional Scd2 ablation in mice; 2) if SCD-CTNNB1 orchestrates in aHSCs, ...